This invention relates to newly identified polynucleotides, polypeptides encoded by such polynucleotides, the use of such polynucleotides and polypeptides, as well as the production of such polynucleotides and polypeptides. More particularly, the polypeptide of the present invention is Human DNA Ligase IV. The invention also relates to inhibiting the action of such polypeptides.
DNA strand interruptions and gaps are generated during replication, repair and recombination. In mammalian cell nuclei, rejoining of such breaks depends on several different DNA polymerases and DNA ligases. The occurrence of three different DNA ligases was established previously by biochemical and immunological characterization of purified enzymes (Tomkinson, A. E., et al., J. Biol. Chem., 266:21728-21735 (1991)). DNA ligases are enzymes that catalyze DNA replication, excision repair and recombinational repair in mammalian cells (Li, J. J. and Kelly, T. J., PNAS, USA, 81:6973-77 (1984) and Wook, R. O. et al., Cell, 53:97-106 (1988)). In bacteria, three DNA ligases, namely DNA Ligase I, DNA Ligase II and DNA Ligase III have been discovered, while in humans all three have been found but only DNA Ligase I has been cloned.
A full-length human cDNA encoding DNA Ligase I has been obtained by functional complementation of a S. cereviasiae cdc9 temperature-sensitive DNA ligase mutant (Barker, D. G., Eur. J. Biochem., 162:659-67 (1987)). The full-length cDNA encodes a 102-kDa protein of 919 amino acid residues. There is no marked sequence homology to other known proteins except for microbial DNA ligases. The active site lysine residue is located at position 568. DNA Ligase I requires magnesium and ATP for activity. The main function of DNA Ligase I is the joining of Okazaki fragments during lagging-strand DNA replication. It also effectively seals single-strand breaks in DNA and joins restriction enzyme DNA fragments with staggered ends. The enzyme is also able to catalyze blunt-end joining of DNA. DNA Ligase I can join oligo (dT) molecules hydrogen-bonded to poly (dA), but the enzyme differs from T4 DNA Ligase in being unable to ligate oligo (dT) with a poly (rA) complementary strand.
Human DNA Ligase II is more firmly associated with the cell nuclei. This enzyme is a labile protein, which is rapidly inactivated at 42xc2x0 C. DNA Ligase II resembles other eukaryotic DNA Ligases in requiring ATP as cofactor, but the enzyme differs from DNA Ligase I in having a much higher association for ATP. DNA Ligase II catalyzes the formation of phosphodiester bonds with an oligo (dT).poly (rA) substrate, but not with an oligo (rA).poly (dT) substrate, so it differs completely from DNA Ligase I in this regard (Arrand, J. E. et al., J. Biol. Chem., 261:9079-82 (1986)).
A recently detected enzyme, which is larger than DNA Ligase II and apparently unrelated to that protein, has been named DNA-Ligase III (Tomkinson, A. E. et al., J. Biol. Chem., 266:21728-35 (1991)). DNA Ligase III resembles DNA Ligase I, and differs from DNA Ligase II, in binding only weakly to hydroxylapatite in having a low affinity, for ATP. DNA Ligase I and III however are not closely related. DNA Ligase III repairs single strand breaks in DNA efficiently, but it is unable to perform either blunt-end joining or AMP-dependent relaxation of supercoiled DNA (Elder, R. H. et al., Eur. J. Biochem., 203:53-58 (1992)).
The mechanism for joining of DNA strand interruptions by DNA ligases has been widely described. The reaction is initiated by the formation of a covalent enzyme-adenylate complex. Mammalian and viral DNA ligases employ ATP as cofactor, whereas bacterial DNA ligases use NAD to generate the adenylyl group. The ATP is cleaved to AMP and pyrophosphate with the adenylyl residue linked by a phosphoramidate bond to the xcex5-amino group of a specific lysine residue at the active site of the protein (Gumport, R. I., et al.., PNAS, 68:2559-63 (1971)). Reactivated AMP residue of the DNA ligase-adenylate intermediate is transferred to the 5xe2x80x2 phosphate terminus of a single strand break in double stranded DNA to generate a covalent DNA-AMP complex with a 5xe2x80x2xe2x80x945xe2x80x2 phosphoanhydride bond. This reaction intermediate has also been isolated for microbial and mammalian DNA ligases, but is more short lived than the adenylylated enzyme. In the final step of DNA ligation, unadenylylated. DNA ligases required for the generation of a phosphodiester bond catalyzes displacement of the AMP residue through attack by the adjacent 3xe2x80x2-hydroxyl group on the adenylylated site.
The polypeptide of the present invention has been putatively identified as a human DNA Ligase IV as a result of amino acid sequence homology.
In accordance with one aspect of the present invention, there are provided novel mature polypeptides which are human DNA Ligase IV, as well as biologically active and diagnostically or therapeutically useful fragments, analogs and derivatives thereof.
In accordance with another aspect of the present invention, there are provided isolated nucleic acid molecules encoding human DNA Ligase IV, including mRNAs, DNAs, cDNAs, genomic DNAs as well as analogs and biologically active and diagnostically or therapeutically useful fragments thereof.
In accordance with yet a further aspect of the present invention, there is provided a process for producing such polypeptides by recombinant techniques comprising culturing recombinant prokaryotic and/or eukaryotic host cells, containing a human DNA Ligase IV nucleic acid sequence, under conditions promoting expression of said protein and subsequent recovery of said protein.
In accordance with yet a further aspect of the present invention, there is provided a process for utilizing such polypeptides, or polynucleotides encoding such polypeptides, for in vitro purposes related to scientific research, synthesis of DNA and manufacture of DNA vectors.
In accordance with another aspect of the present invention there is provided a method of treating conditions which are related to insufficient human DNA Ligase IV activity via gene therapy comprising inserting the DNA Ligase IV gene into a patient""s cells either in vivo or ex vivo. The gene is expressed in transduced cells and as a result, the protein encoded by the gene may be used therapeutically, for example, to prevent disorders associated with defects in DNA, for example, abnormal cellular proliferation, for example tumors, to treat severe immunosuppression, stunted growth and lymphoma, as well as cellular hypersensitivity to DNA-damaging agents.
In accordance with yet a further aspect of the present invention, there is also provided nucleic acid probes comprising nucleic acid molecules of sufficient length to specifically hybridize to human sequences which may be used diagnostically to detect a mutation in the gene encoding DNA Ligase IV.
In accordance with yet another aspect of the present invention, there are provided antagonists to such polypeptides, which may be manufactured intracellularly or administered through gene therapy to inhibit the action of such polypeptides, for example, to target and destroy undesired cells, e.g., cancer cells.
These and other aspects of the present invention should be apparent to those skilled in the art from the teachings herein.